The present invention relates generally to the treatment of viral infections using lipid derivatives of antiviral nucleoside analogues. More particularly, the present invention relates to lipid, and especially phospholipid, derivatives of modified antiviral nucleoside analogues which can be integrated into the structure of liposomes, thereby forming a more stable liposomal complex which can deliver greater amounts of drugs to target cells with less toxicity.
The publications and other reference materials referred to herein are hereby incorporated by reference, and are listed for convenience in the bibliography appended at the end of this specification.
There has been a great deal of interest in recent years in the use of nucleoside analogues to treat viral infections. A nucleoside consists of a pyrimidine or purine base which is linked to ribose, a five-carbon sugar having a cyclic structure. The antiviral nucleoside analogues closely resemble natural nucleosides and are designed to inhibit viral functions by preventing the synthesis of new DNA or RNA. Nucleosides are enzymatically assembled into DNA or RNA.
During DNA synthesis, free nucleoside triphosphates (nucleosides with three phosphate groups attached) react with the end of a growing DNA chain. The reaction involves the linking of the phosphate group at the 5xe2x80x2 position on the incoming nucleoside triphosphate with the hydroxyl group at the 3xe2x80x2 position of the sugar ring on the end of the forming DNA chain. The other two phosphate groups are freed during the reaction, thereby resulting in the addition of a nucleotide to the DNA chain.
Nucleoside analogues are compounds which mimic the naturally occurring nucleosides sufficiently so that they are able to participate in viral DNA synthesis. However, the antiviral nucleoside analogues have strategically located differences in chemical structure which inhibit viral enzymes such as reverse transcriptase or which prevent further DNA synthesis once the analogue has been attached to the growing DNA chain.
Dideoxynucleosides are antiviral compounds that lack the hydroxyl groups normally present at the second and third position of ribose. When a dideoxynucleoside is incorporated into a growing DNA chain, the absence of the 3-OH group on its ribose group makes it impossible to attach another nucleotide and the chain is terminated. Dideoxynucleosides are particularly useful in treating retroviral infections where viral replication requires the transcription of viral RNA into DNA by viral reverse transcriptase. Other nucleoside analogues include deoxynucleosides and nucleosides analogues having only a fragment of ribose or other pentose connected to the base molecule.
Acquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV). HIV infects cells bearing the CD4 (T4) surface antigen, such as CD4+ helper lymphocytes, CD4+ monocytes and macrophages and certain other CD4+ cell types. The HIV infection of CD4+ lymphocytes results in cytolysis and cell death which contributes to the immunodeficiency of AIDS; however, CD4+ monocytes and macrophages may not be greatly harmed by the virus. Viral replication in these cells appears to be more prolonged and less cytotoxic than in lymphocytes, and as a result, monocytes and macrophages represent important reservoirs of HIV infection. It has recently been discovered that macrophages may serve as reservoirs of HIV infection even in certain AIDS patients who test negative for the presence of HIV antibodies. No effective cure is available for AIDS, although dideoxynucleosides have been shown to prolong life and to reduce the incidence of certain fatal infections associated with AIDS.
Certain monocyte-derived macrophages, when infected with some strains of HIV, have been found to be resistant to treatment with dideoxycytidine, azidothymidine, and other dideoxynucleosides in vitro as shown by Richman, et al. (1). The resistance may be due in part to the low levels of dideoxynucleoside kinase which result in a reduced ability to phosphorylate AZT, ddC or ddA. Clearly, it would be useful to have more effective ways of delivering large amounts of effective antiviral compounds to macrophages infected with HIV or other viruses and other cells having viral infections. It would also be useful to have more effective ways of delivering antiviral compounds which not only increase their potency but prolong their efficacy.
Dideoxynucleoside analogues such as AZT are the most potent agents currently known for treating AIDS, but in a recent human trial, serious toxicity was noted, evidenced by anemia (24%) and granulocytopenia (16%) (2,3). It is desirable, therefore, to provide a means for administering AZT and other dideoxynucleosides in a manner such that the toxic side effects of these drugs are reduced. Further, it is desirable to provide selective targeting of the dideoxynucleoside to monocyte/macrophages to enhance the efficiency of the drug against viral infection in this group of cells. One way to do this is to take advantage of the uptake of liposomes by macrophages.
In 1965, Alex Bangham and coworkers discovered that dried films of phosphatidylcholine spontaneously formed closed bimolecular leaflet vesicles upon hydration (4). Eventually, these structures came to be known as liposomes.
A number of uses for liposomes have been proposed in medicine. Some of these uses are as carriers to deliver therapeutic agents to target organs. The agents are encapsulated during the process of liposome formation and released in vivo when liposomes fuse with the lipids of cell surface membrane. Liposomes provide a means of delivering higher concentrations of therapeutic agents to target organs. Further, since liposomal delivery focuses therapy at the site of liposome uptake, it reduces toxic side effects.
For example, liposomal antimonial drugs are several hundred-fold more effective than the free drug in treating leishmaniasis as shown independently by Black and Watson (5) and Alving, et al. (6). Liposome-entrapped amphotericin B appears to be more effective than the free drug in treating immunosuppressed patients with systemic fungal disease (7). Other uses for liposome encapsulation include restriction of doxorubicin toxicity (8) and diminution of aminoglycoside toxicity (9).
As previously mentioned, it is now thought that macrophages are an important reservoir of HIV infection (10, 11). Macrophages are also a primary site of liposome uptake (12, 13). Accordingly, it would be desirable to utilize liposomes to enhance the effectiveness of antiviral nucleoside analogues in treating AIDS and other viral infections.
The use of liposomes to deliver phosphorylated dideoxynucleoside to AIDS infected cells which have become resistant to therapy has been proposed in order to bypass the low dideoxynucleoside kinase levels.
Attempts have also been made to incorporate nucleoside analogues, such as iododeoxyuridine (IUDR), acylovir (ACV) and ribavirin into liposomes for treating diseases other than AIDS. However, these attempts have not been entirely satisfactory because these relatively small water soluble nucleoside analogues tend to leak out of the liposome rapidly (14, 15), resulting in decreased targeting effectiveness. Other disadvantages include the tendency to leak out of liposomes in the presence of serum, difficulties in liposome formulation and stability, low degree of liposomal loading, and hydrolysis of liposomal dideoxynucleoside phosphates when exposed to acid hydrolases after cellular uptake of the liposomes.
Attempts have also been made to combine nucleoside analogues, such as arabinofuranosylcytosine (ara-C) and arabinofuranosyladenine (ara-A), with phospholipids in order to enhance their catabolic stability as chemotherapeutic agents in the treatment of various types of cancer (16). The resulting agents showed a decreased toxicity and increased stability over the unincorporated nucleoside analogues. However, the resulting agents exhibited poor cellular uptake (16) and poor drug absorption (17).
In order to use nucleoside analogues incorporated into liposomes for treating viral infections more effectively, it is desirable to increase the stability of the association between the liposome and the nucleoside analogue.
In order to further enhance the effectiveness of these antiviral liposomes, it would be desirable to target the liposomes to infected cells or sites of infection. Greater specificity in liposomal delivery may be obtained by incorporating monoclonal antibodies or other ligands into the liposomes. Such ligands will target the liposomes to sites of liposome uptake capable of binding the ligands. Two different approaches for incorporating antibodies into liposomes to create immunoliposomes have been described: that of Huang and coworkers (18) involving the synthesis of palmitoyl antibody, and that of Leserman, et al. (19) involving the linkage of thiolated antibody to liposome-incorporated phosphatidylethanolamine (PE).
The methods disclosed here apply not only to dideoxynucleosides used in the treatment of AIDS and other retroviral diseases, but also to the use of antiviral nucleosides in the treatment of diseases caused by other viruses, such as herpes simplex virus (HSV), human herpes virus 6, cytomegalovirus (CMV), hepatitis B virus, Epstein-Barr virus (EBV), and varicella zoster virus (VZV). Thus, the term xe2x80x9cnucleoside analoguesxe2x80x9d is used herein to refer to compounds that can inhibit viral replication at various steps, including inhibition of viral reverse transcriptase or which can be incorporated into viral DNA or RNA, where they exhibit a chain-terminating function.
The invention provides a composition for use in treating viral infections, including HIV (AIDS), herpes simplex virus (HSV), human herpes virus 6, cytomegalovirus (CMV), hepatitis B virus, Epstein-Barr virus (EBV), and varicella zoster virus (VZV). The composition may contain, in addition to a pharmaceutically acceptable carrier, a lipophilic antiviral agent prepared by chemically linking an antiviral nucleoside analogue to at least one lipid species. The antiviral nucleoside analogue may be linked to the lipid through a monophosphate, diphosphate or triphosphate group. The invention, further, provides a method for incorporating such lipid derivatives of antiviral agents into liposomes for improved delivery of the antiviral agent. A liposome comprises a relatively spherical bilayer which is comprised wholly or in part of the above-described lipid derivatives of antiviral agents. The liposome may also contain pharmacologically inactive lipids. Further, the liposome may contain a ligand, such as a monoclonal antibody to a viral binding site (such as CD4), or other binding protein. Such a ligand provides additional specificity in the delivery site of the antiviral agent. The invention provides a method for incorporating such ligands into antiviral liposomes.
In one preferred embodiment, the compound is a phosphatidyldideoxynucleoside or a dideoxynucleoside diphosphate diglyceride. In another, the lipid species may comprise at least one acyl ester, ether, or vinyl ether group of glycerol-phosphate. Phosphatidic acids having at least one acyl ester, ether, or vinyl ether group may also serve as a favored lipid species.
In another embodiment, the nucleoside analogue is a purine or pyrimidine linked through a xcex2-N-glycosyl bond to a pentose residue that lacks at least one of the 2xe2x80x2 or 3xe2x80x2 carbons, but retains the 5xe2x80x2 carbon, and the phosphate group is bound to the 5xe2x80x2 carbon (i.e., what would have been the 5xe2x80x2 carbon in a complete pentose moiety). In another embodiment of the invention, the lipid species is an N-acyl sphingosine.
In some preferred embodiments, the acyl or alkyl groups of the lipid species, of whatever linkage, as for example ester, ether or vinyl ether, comprise 2 to 24 carbon atoms. In one variation, at least one of the acyl or alkyl groups is saturated. In another, at least one of the acyl or alkyl groups has up to six double bonds. In yet another embodiment, an acyl or alkyl group may be attached directly by ester or alkyl linkage to the 5xe2x80x2-hydroxyl of the nucleoside.
In still another, the lipid moiety is a glyceride and the glyceride has two acyl groups that are the same or different. In still another embodiment of the invention, the lipid species is a fatty alcohol residue which is joined to a phosphate linking group through an ester bond. The compound may advantageously have from one to three phosphate groups, and at least one fatty alcohol ester, and may have two or more fatty alcohol residues that are the same or different in structure. These fatty alcohols are preferably linked to the terminal phosphate group of the compound.
Moreover, the invention includes a composition wherein, in addition to the compound, the liposome further comprises phospholipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol and sphingomyelin.
In one embodiment of the invention, the percentage of antiviral agent is 0.01 to 100 percent by weight of the liposome.
In another embodiment, the liposome further comprises a ligand bound to a lipid substrate. The ligand may be an antibody, such as a monoclonal antibody to a viral antigen. The viral antigen could be gp41 or gp110 of HIV, or could be any other suitable viral antigen. In one embodiment, the ligand is CD4 receptor protein, or CD4 protein itself. Alternatively, the ligand is an antibody to CD4 or a protein or other substance that binds CD4.
The invention also contemplates a composition for use in treating viral and retroviral infections, comprising a liposome formed at least in part of an lipophilic antiviral agent, the agent comprising a nucleoside analogue having a base and a pentose residue with at least one lipid species attached to the nucleoside analogue through a monophosphate, diphosphate or triphosphate linking group at the 5xe2x80x2 hydroxyl of the pentose residue of the nucleoside analogue, and a pharmaceutically acceptable carrier therefore.
Thus, there is provided a composition having antiviral properties, comprising an antiviral nucleoside analogue having a base portion comprising a substituted or unsubstituted purine or pyrimidine, and a sugar portion comprising a pentose residue, and a lipid moiety linked to the pentose residue, with the proviso that the composition is in the form of a liposome when the pentose residue is ribose and the base portion is cytosine, and when the pentose residue is arabinofuranose and the base portion is cytosine or adenine. In one embodiment, the nucleoside analogue is a nitrogenous base which is a purine, pyrimidine, or a derivative thereof, and the pentose residue is a 2xe2x80x2,3xe2x80x2-dideoxy, 2xe2x80x2,3xe2x80x2-didehydro, azido or halo derivative of ribose, or an acyclic hydroxylated fragment of ribose. The pentose residue may thus be a 2xe2x80x2,3xe2x80x2-dideoxyribose, and the nucleoside analogue may be 2xe2x80x2,3xe2x80x2-dideoxycytidine, 2xe2x80x2,3xe2x80x2-dideoxythymidine, 2xe2x80x2,3xe2x80x2-dideoxyguanosine, 2xe2x80x2,3xe2x80x2-dideoxyadenosine, 2xe2x80x2,3xe2x80x2-dideoxyinosine, or 2,6 diaminopurine, 2xe2x80x2,3xe2x80x2-dideoxyriboside.
In another embodiment, the pentose residue is a 2xe2x80x2,3xe2x80x2-didehydroribose and the nucleoside is 2xe2x80x2,3xe2x80x2-didehydrothymidine, 2xe2x80x2,3xe2x80x2-didehydrocytidine carbocyclic, or 2xe2x80x2,3xe2x80x2-didehydroguanosine. In still another embodiment, the pentose residue is an azide derivative of ribose, and the nucleoside is 3xe2x80x2-azido-3xe2x80x2-deoxythymidine, 3xe2x80x2-azido-3xe2x80x2-deoxyguanosine, or 2,6-diaminopurine-3-azido-2xe2x80x2,3xe2x80x2dideoxyriboside.
In still another embodiment of the invention, the pentose residue is a halo derivative of ribose and the nucleoside is 3xe2x80x2-fluoro-3xe2x80x2-deoxythymidine, 3xe2x80x2-fluoro-2xe2x80x2,3xe2x80x2-dideoxyguanosine, 2xe2x80x2,3xe2x80x2-dideoxy-2xe2x80x2-fluoro-ara-adenosine, or 2,6-diaminopurine-3xe2x80x2-fluoro-2xe2x80x2,3xe2x80x2-dideoxyriboside. The invention also includes halo derivatives of the purine or pyrimidine rings, such as, for example, 2-chloro-deoxyadenosine. Alternatively, the pentose residue is an acyclic hydroxylated fragment of ribose, and the nucleoside is 9-(4,-hydroxy-1xe2x80x2,2xe2x80x2-butadienyl) adenine, 3-(4,-hydroxy-1xe2x80x2,2xe2x80x2-butadienyl) cytosine, 9-(2-phosphonylmethoxyethyl)adenine or phosphonomethoxydiaminopurine.
In accordance with another aspect of the invention, the nucleoside analogue is acyclovir, gancyclovir, 1-(2xe2x80x2-deoxy-2xe2x80x2-fluoro-1-xcex2-D-arabinofuranosyl)-5-iodocytosine (FIAC) or 1(2xe2x80x2-deoxy-2xe2x80x2-fluoro-1-xcex2-D-arabinofuranosyl)-5-iodouracil (FIAU).
In all of the foregoing compositions, a monophosphate, diphosphate, or triphosphate linking group may be provided between the 5xe2x80x2 position of the pentose residue and the lipid species. Alternatively, there may be an aliphatic bridge comprising two functional groups and having from 0 to 10 carbon atoms between the functional groups, the bridge joining the lipid and the pentose residue. In still further embodiments of the invention, the lipid species is a fatty acid, a monoacylglycerol, a diacylglycerol, or a phospholipid. The phospholipid may have a head group comprising a sugar or a polyhydric alcohol. Specific examples of phospholipids include bis(diacylglycero)-phosphate and diphosphatidylglycerol. Other examples of lipid species include D,L-2,3-diacyloxypropyl-(dimethyl)-beta-hydroxyethyl ammonium groups.
In accordance with another aspect of the present invention, the lipid species comprises from 1 to 4 fatty acid moieties, each the moiety comprising from 2 to 24 carbon atoms. Advantageously, at least one fatty acid moiety of the lipid species is unsaturated, and has from 1 to 6 double bonds.
Particular examples of these compositions include 3-phosphonomethoxyethyl-2,6-diaminopurine; 1,2-diacylglycerophospho-5xe2x80x2-(2xe2x80x2,3xe2x80x2-dideoxy)thymidine.
Specific compositions are provided having the formula:
(L)mxe2x80x94(W)nxe2x80x94Axe2x80x94Qxe2x80x94Z
wherein
Z is the base portion of the nucleoside analogue, Q is the pentose residue, A is O, C, or S, W is phosphate, n=0 to 3, and L is a lipid moiety wherein m=1 to 5, and wherein each L is linked directly to a W except when n=0, in which case each L is linked directly to A.
Also included are compositions having the formula: 
wherein Z is the substituted or unsubstituted purine or pyrimidine group of the nucleoside analogue,
Q is the pentose residue,
W is phosphate, A is O, C, or S, L1 is (CH2xe2x80x94CHOHxe2x80x94CH2), and
L is a lipid moiety.
In one embodiment of the invention, with reference to the foregoing formulas, each L is independently selected from the group consisting of R, 
wherein R, R1 and R2 are independently C2 to C24 aliphatic groups and wherein R, R1 and R2 independently have from 0 to 6 sites of unsaturation, and have the structure CH3xe2x80x94(CH2)axe2x80x94(CHxe2x95x90CHxe2x80x94CH2)bxe2x80x94(CH2)cxe2x80x94Y
wherein the sum of a and c is from 1 to 23, and b is 0 to 6, and wherein Y is C(O)Oxe2x80x94, Cxe2x80x94Oxe2x80x94, Cxe2x95x90Cxe2x80x94Oxe2x80x94, C(O)Sxe2x80x94, Cxe2x80x94Sxe2x80x94, or Cxe2x95x90Cxe2x80x94Sxe2x80x94.
In one embodiment of the foregoing compositions, the pentose residue comprises ribose, dideoxyribose, didehydroribose, or an azido or halosubstituted ribose, attached at the 9 position of the purine or at the 1 position of the pyrimidine.
The present invention also provides a method for synthesizing a lipid derivative of an antiviral nucleoside, comprising the step of reacting an antiviral nucleoside, having a ribose hydroxyl group, with a phospholipid in the presence of a coupling reagent whereby the nucleoside is joined to the phospholipid by a phosphate bond at the position of the ribose hydroxyl group. In one preferred embodiment, the phospholipid is a diacyl phosphate. In another, the phospholipid is a phosphatidic acid or a ceramide. Also provided herein is a method of synthesizing a lipid derivative of an antiviral nucleoside, comprising the steps of reacting an antiviral nucleoside monophosphate with a reagent HL, wherein L represents a leaving group, to form a nucleoside PO4-L, reacting the nucleoside PO4-L with a phosphatidic acid to bind the acid to the nucleoside through a pyrophosphate bond. In one variation of the method, the nucleoside monophosphate is AZT 5xe2x80x2-monophosphate.
Still a further method provided by the present invention is a method of synthesizing a glyceride derivative of a nucleoside analogue, comprising the step of joining a monoglyceride or diglyceride and an antiviral nucleoside monophosphate with a coupling agent in the presence of a basic catalyst. In one embodiment, the glyceride is 1-O-stearoylglycerol and the nucleoside is AZT monophosphate.
Also a part of the present invention is a method for preparing a suspension of liposomes for use in treating viral and retroviral infections in a mammal, comprising providing a lipophilic antiviral agent comprising at least one lipid species attached to a nucleoside analogue through a monophosphate, diphosphate or triphosphate linking group at the 5xe2x80x2 position of the pentose residue of the nucleoside, combining the lipophilic antiviral agent and a pharmacologically acceptable aqueous solvent to form a mixture, and forming liposomes from the lipophilic antiviral agent. The liposomes may be formed, for example, by sonication, extrusion or microfluidization. In one preferred embodiment, the combining step further comprises including in the combination a pharmacologically inactive lipophilic lipid. This inactive lipid can be, for example, a phosphatidylethanolamine, a sphingolipid, a sterol or a glycerophosphatide. The method also may include treating the liposomes with thio-antibodies to produce immunoliposomes, or including in the combination an lipophilic lipid which is, in part, comprised of a ligand. Thus, the liposome may include a ligand bound to a lipid substrate.
In addition, the invention includes a method for treating retroviral and viral infections in a mammal, such as a human, by administering a sufficient quantity of the antiviral nucleoside analogues described herein to deliver a therapeutic dose of the antiviral agent to the mammal. In a preferred embodiment, the method is used to treat retroviral and viral infections in a mammal, wherein the retrovirus has become resistant to therapy with conventional forms of an antiviral agent. The present invention also includes a method for treatment of patients having strains of HIV that have developed resistance to AZT or reduced sensitivity to AZT, comprising the step of administering a composition of the present invention to such patient in an effective, retrovirus-inhibiting dosage. Also included in the present invention is a method for treating a viral infection in a mammal, comprising the step of administering an effective amount of a composition as described herein to a mammal. The infection may be a herpes simplex infection, and the composition may be phosphatidylacyclovir. Alternatively, the virus may be HIV retrovirus, and the composition may be 5xe2x80x2-palmitoylAZT. The method includes use where the retrovirus is a strain of HIV that has developed resistance to a nucleoside analogue.
Also disclosed herein is a method for prolonging the antiviral effect of a nucleoside analogue in a mammal, comprising administering the nucleoside analogue to the mammal in the form of the nucleoside-lipid derivatives disclosed herein. Also disclosed is a method for avoiding or overcoming resistance of the retrovirus to nucleoside analogues through administering the analogue in the form of the lipid derivative compositions disclosed herein.
Finally, the present invention includes use of these compositions in the preparation of a medicament for treatment of a human viral infection.
Liposomal delivery of antiretroviral and antiviral drugs results in higher dosing of macrophage and monocyte cells which take up liposomes readily. The unique advantages of the present invention are that the lipid derivatives of the antiviral nucleosides are incorporated predominantly into the phospholipid layer of the liposome rather than in the aqueous compartment. This allows larger quantities of antiviral analogue to be incorporated in liposomes than is the case when water soluble phosphate esters of the nucleosides are used. Complete incorporation of the antiviral derivative into liposomes will be obtained, thus improving both the drug to lipid ratio and the efficiency of formulation. Further, there will be no leakage of the antiviral lipid analogues from the liposome during storage. Finally, liposomal therapy using these compositions allows larger amounts of antiviral compound to be delivered to the infected macrophage and monocyte cells. Therapy with liposomal compositions containing site specific ligands allows still greater amounts of antiviral compounds to be delivered with increased specificity.
Another novel advantage of this invention is that each class of lipid derivatives of antiviral nucleosides disclosed below is believed to give rise directly to antiviral phosphorylated or non-phosphorylated nucleosides upon cellular metabolism.
A further advantage of this invention is that the novel lipid derivatives are incorporated into the cell, protecting the cell for prolonged periods of time, up to or exceeding 48 hours after the drug is removed.
These and other advantages and features of the present invention will become more fully apparent from the following description and appended claims.